Traumatic brain injury (TBI) is characterized by sudden physical damage to the brain. It is caused by many factors including warfare, automobile accidents, sports injuries, violent crimes, household accidents, child abuse or by an object passing through the skull, for example gun shot wounds. The physical, behavioral, or mental changes that may result from TBI depend on the areas of the brain that are injured. Most injuries cause focal brain damage, damage confined to a small area of the brain. The focal damage is most often at the point where the head hits an object or where an object, such as a bullet, enters the brain.
In warfare, TBI can be presented as penetrating wounds from high-velocity bullets and debris from explosions from improvised explosive devices (IEDs), including road side mines, or as diffuse brain injury due to blasts caused by IEDs. Similar injuries are suffered by civilians who are victims of terrorist bomb attacks, typically using IEDs. Due to the improved care at the frontline and speedy response to terrorist attacks, a larger proportion of victims suffering can be rescued. It was found, however, that despite continued efforts to hasten evacuation from the field and improve the management of explosion-related head trauma, the outcome of treatment is still far from satisfactory.
While treatment of head wounds immediately after injury has improved steadily in the past few decades, lingering effects such as disabilities are still likely after moderate and severe TBI [Kluger Y, et al. J Am Coll Surg. 2004;199:875-879]. It is now well understood that the primary injury of TBI is followed for hours and days by a process of secondary injury [Gaetz M. et al. Clin Neurophysiol. 2004;115:4-18; Sullivan PG, et al. J Neurosci Res. 2005;79:231-239]. Major factors contributing to this second wave of brain damage, include: excitatory amino acids such as glutamate, Ca++ homeostasis, and reactive oxygen species (ROS) [Kluger et al., Gaetz et al]. Mitochondria are one of the cell organelles affected by secondary brain injury, and in particular the mitochondrial permeability transition pore (MPTP) appears to take a central role among the factors leading to neuronal cell death with secondary brain injury [Kluger et al., Gaetz et al]. This suggests that targeting the MPTP, immediately after sustained brain injury, would have significant therapeutic implications for TBI.
The MPTP consists of three components which are identical to those described for the peripheral-type benzodiazepine receptor (PBR). PBR are present in peripheral tissues, and also in brain cells. The MPTP/PBR-complex, which is located in the mitochondrial membrane, is composed of three protein components: the 18-kDa isoquinoline binding protein (IBP), the 32-kDa voltage dependent anion channel (VDAC), and the 30-kDa adenine nucleotide transporter (ANT) (FIG. 1). In situ, the PBR/MPTP-complex is composed of several 18-kDa IBP components for each VDAC molecule (see FIG. 1). Functions for this receptor complex include involvement in apoptosis, ischemia, regulation of the mitochondrial membrane potential, mitochondrial respiration, steroidogenesis, immune responses of the cardiovascular system, cell proliferation, and cancer.
The PBR-complex binds specifically with the benzodiazepine Ro5 4864 (4′-chlorodiazepam) and the isoquinoline carboxamide derivative PK 11195 (FIG. 2), but not the central benzodiazepine receptor ligand clonazepam [Veenman L et al. Pharmacol. Ther. 2005;[E-pub ahead of print]. Later, to these classical PBR ligands, a new class of compounds, the 2 aryl-3-indoleacetamides (FGIN-1), was found to potently (with nM affinity) and selectively bind to the PBR-complex [Veenman L et al 2005]. A representative example is FGIN-1-27 (with a Ki=4.4±0.1 nM, as measured by the displacement of [3H]Ro5 4864 (FIG. 2).
Some of the applicants of the present invention and others have demonstrated that PBR levels increase in the brain after brain damage, including traumatic brain damage and epilepsy. It has also been shown that knockdown of PBR expression and PBR ligands can prevent cell death of glial cells, including apoptosis (FIG. 3A). Moreover, some of the applicants of the present invention have shown that classical PBR ligands can prevent neurodegeneration typically caused by excitatory amino acids [Veenman L et al. J. Neurochem. 2002;80:917-927]. In particular, it was found that treatment with the PBR ligand, PK 11195, prevented the effects of kainic acid injections, including modulation of PBR composition and prevention of seizures [Veenman L et al. 2002]. This protective effect of PBR ligands against neurodegeneration was later confirmed in other studies [Ryu J. K et al. Neurobiol. Dis. 20:550-561, Veiga S et al. J Neurosci Res. Apr. 1, 2005;80(1):129-37]. All of the above studies suggest that PBR may form prime targets to treat and prevent secondary brain injury following TBI, including their consequences such as disability and epilepsy. PBR ligands may modulate apoptosis by causing changes in mitochondrial membrane potential via opening of the MPTP i.e. the VDAC component of the PBR-complex. This precedes and initiates a cascade including cytochrome c and caspases 9 and 3 leading to apoptosis.
It has also been found using genetic manipulation studies, that knockdown of the IBP component of the PBR-complex in the C6 glial cell line completely prevented apoptosis induced by the PBR ligand, FGIN-1-27 (FIG. 3A), as well as by non-PBR ligands, such as the anticancer agent, ErPC (FIG. 3B). Furthermore, knockdown of the IBP component of the PBR-complex reduced basal levels of apoptosis. Moreover, knockdown of the IBP component of the PBR-complex in the C6 glial cell line completely prevented apoptosis caused by a major contributor of neuronal cell death, the excitatory amino acid glutamate, and by one of the causative agents for the neuronal cell death in the neurodegenerative disease of Alzheimer, Abeta(1-42). This indicates that the IBP component of the PBR-complex plays an essential role in the induction of apoptosis, which is one of cellular processes leading the neuronal cell death. Substantiating this finding, the applicants of the present invention have shown that opening of the MPTP component of the MPTP/PBR complex by the ErPC requires IBP (FIG. 4). This indicates that IBP can serve to modulate the activity of VDAC and ANT i.e. the MPTP, and consequently modulate the mitochondrial apoptosis pathway.
Additional data resulting from knockdown of the ANT2 component of the MPTP/PBR-complex showed that this component also is required to induce apoptosis by ErPC. Furthermore, it was found that overexpression of the VDAC component of the MPTP/PBR-complex reduced the mitochondrial membrane potential. Overall, the data show that the major components to the MPTP/PBR-complex appear to play important roles in the induction of apoptosis.
It has also been shown by the applicants of the present invention, that the classical PBR ligands, PK 11195 and Ro5 4864, can prevent apoptotic cell death induced by strong pro-apoptotic agents [Kugler W et al. Abstracts of the 14th Annual Meeting of the Israel Society for Neuroscience, and the Joint Germany-Israel Meeting, Eilat, Israel, 2005;16:S38 ]. Thus, their effects are similar to those of knockdown of PBR components. Therefore, these PBR ligands can be considered weak or strong antagonists to PBR. In contrast, FGIN-1-27 can be considered to be an agonist [Veenman L et al. 2005]. However, pro-apoptotic effects of PK 11195 and Ro5 4864 are also reported, in particular at high concentrations, which are not due to their interaction with PBR, but may be caused by their effects on plasma membrane calcium channels [Veenman L et al. 2005]. Given the important effects on the life essential functions of PBR, several new PBR ligands have been developed [Veenman L et al. 2005]. Extensive structure-activity relationship studies of known PBR ligands revealed that their effects are affected by very slight structural modifications [Veenman L et al. 2005]. Based on the results of these structure-activity relationship studies, several different structures were elaborated such as 2-aryl substituted benzofuran-3-acetamide derivatives and also several isoquinolines [Benavides J et al. J Neurochem 1983;41:1744-1750], imidazopyridines [Langer S Z et al. Pharmacologicol Biochein Behav 1988;29:763-766], and pyrrolobenzoxazepine (1 in FIG. 5) [Campiani G, et al. J Med Chem 1996;39:3435-3450]. According to these models, the receptor binding site provides a hydrogen bond-donating function (H1) and two lipophilic pockets (L1 and L2) for all these ligands. The presence of an additional lipophilic region (L3) within the receptor has been deduced from these analyses (FIG. 4).
Although the molecules mentioned above (Ro5 4864, PK 11195, FGIN-1-27 and pyrrolobenzoxazepine) seem to be structurally very different, closer inspection reveals a number of common structural features which may be necessary for the efficacy of these ligands. These common features are listed below and presented in FIGS. 5 and 6:                1) The central core, designated to L1 in FIG. 5, (central carbocycle, C2 to C6) always has an additional aromatic ring (C7 to C10 or C11) attached to it.        2) An aryl ring, designated to L2 in FIG. 5, which may sometimes have a halogen or a second aromatic ring (naphtyl) attached in C4′or C2′(see C1′to C6′ in FIG. 6 for all the compounds).        3) This aryl ring is always linked to a double bond designated to L1 (FIG. 5) (C5-C4 for Ro5 4864 and PK 11195 and C5-C3 for FGIN-1-27 and pyrrolobenzoxazepine) (FIG. 6), while C4 in this aryl ring is always substituted by a heteroatom (nitrogen in Ro5 4864, PK 11195 and FGIN-1-27, and oxygen in pyrrolobenzoxazepine).        4) All carbocycles are planar (FIG. 6) and therefore no stereogenic centers are needed for the recognition.        5) An amide link (designated to L3 in FIG. 5) is always present in the carbon skeleton either included in the carbocycle, as in Ro5 4864, or outside the carbocycle in PK 11195, FGIN-1-27 and pyrrolobenzoxazepine (FIG. 6).        6) The same amide link also provides for the hydrogen bond-donating function (H1, in FIG. 5), responsible for the hydrogen bond formation inside the IBP binding site.        
U.S. Pat. No. 6,765,006 discloses quinazolines and other heterocycles which are antagonists or positive modulators of AMPA receptors, and the use thereof for treating, preventing or ameliorating neuronal loss or treating or ameliorating neurodegenerative diseases.
A need in the art exists to develop novel agents which bind more selectively to PBR than current PBR ligands and which prevent brain cell death, including apoptosis, as it occurs due to TBI and/or neurodegenerative diseases.